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An investigation of the mechanism of UDP-glucose dehydrogenase Penney, Lisa C.


UDP-glucose dehydrogenase (UDPGluDH) is an NAD⁺-dependent enzyme that catalyzes the two-fold oxidation of UDP-glucose to UDP-glucuronic acid. This enzyme is interesting in that its single active site carries out two sequential oxidations, whereas the majority of NAD⁺ - dependent dehydrogenases catalyze only a single oxidation step. Bacterial UDPGluDH is necessary for the formation of an antiphagocytic capsule that protects many virulent strains from the host's immune system. It has been determined that the enzymatic mechanism proceeds through covalent catalysis using an active site cysteine residue. Hydrolysis of the resulting thioester intermediate generates the UDP-glucuronic acid product. The structure of wild-type UDPGluDH has been previously elucidated in our laboratory. This allowed residues that may be important in the enzyme's catalytic mechanism to be identified and several mutant forms of the enzyme to be constructed. Three of these mutants - Thrll8Ala, Glul41Gln, and Glul45Gln - have been purified to homogeneity and their K[sub m] and K[sub cat] values have been determined. It was found that the k[sub cat] value of the Thr118Ala UDPGluDH was reduced by a factor of 100 when compared to that of the wild-type enzyme. When this mutant enzyme was incubated with UDP-(6,6-di-²H)glucose, the value of k[sub H]/k[sub D] was determined to be 1.9 ± 0.1, indicating that a C-H bond is broken in the enzyme's rate-determining step. This differs from previously obtained results using the wild-type enzyme, in which it was found that the hydride transfer steps were not rate-limiting. Thus, it appears that Thr118 plays a more important role in one or both of the hydride transfer steps of the enzymatic mechanism than in the hydrolysis of the thioester intermediate.

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